Acute myeloid leukemia (AML) represents a significant unmet medical need. It is a hematological malignancy characterized by a block in differentiation and aberrant proliferation of the myeloid lineage of hematopoietic progenitor cells. There are approximately 13,000 new cases and 9,000 deaths per year in the United States. The survival rate is 25-70% in patients younger than 60 years and 5-15% in older patients, with worse outcomes in patients with poor risk cytogenetics. Current standard of care treatment is daunorubicin and cytarabine chemotherapy with induction and consolidation phases. Bone marrow stem cell transplant is also used for treating AML in younger patients.
Cyclin-dependent kinases (CDKs) are a family of serine/threonine protein kinases playing important cellular functions. The cyclins are the regulatory subunits that activate the catalytic CDKs. CDK1/Cyclin B1, CDK2/Cyclin A, CDK2/Cyclin E, CDK4/Cyclin D, CDK6/Cyclin D are critical regulators of cell cycle progression. CDKs also regulate transcription, DNA repair, differentiation, senescence and apoptosis (Morgan, D. O., Annu. Rev. Cell. Dev. Biol., 13:261-291 (1997)).
Small molecule inhibitors of CDKs have been developed to treat cancer (de Carcer, G. et al., Curr. Med. Chem., 14:969-85 (2007)). A large amount of genetic evidence supports that CDKs, their substrates or regulators have been shown to be associated with many human cancers (Malumbres, M. et al, Nature Rev. Cancer, 1:222-231 (2001)). Endogenous protein inhibitors of CDKs including p16, p21 and p27 inhibit CDK activity and their overexpression results in cell cycle arrest and inhibition of tumor growth in preclinical models (Kamb, A., Curr. Top. Microbiolo. Immunol., 227:139-148 (1998)).
Small molecule inhibitors of CDKs may also be used to treat variety of other diseases that result from aberrant cell proliferation, including cardiovascular disorders, renal diseases, certain infectious diseases and autoimmune diseases. Cell proliferation pathways including genes involved in the cell cycle G1 and S phase checkpoint (p53, pRb, p15, p16, and Cyclins A, D, E, CDK 2 and CDK4) have been associated with plaque progression, stenosis and restenosis after angioplasty. Over-expression of the CDK inhibitor protein p21 has been shown to inhibit vascular smooth muscle proliferation and intimal hyperplasia following angioplasty (Chang, M. W. et al., J. Clin. Invest., 96:2260 (1995); Yang, Z-Y. et al., Proc. Natl. Acad. Sci. (USA) 93:9905 (1996)). A small molecule CDK2 inhibitor CVT-313 (Ki=95 nM) was shown to cause significant inhibition of neointima formation in animal models (Brooks, E. E. et al., J. Biol. Chem., 272:29207-29211 (1997)). Disregulation of cell cycle has been associated with polycystic kidney diseases, which are characterized by the growth of fluid-filled cysts in renal tubules. Treatment with small molecule inhibitors of CDKs yielded effective arrest of cystic disease in mouse models (Bukanov, N. O., et al., Nature, 4444:949-952 (2006)). Infection by a variety of infectious agents, including fungi, protozoan parasites such as Plasmodium falciparum, and DNA and RNA viruses may be treated with CDK inhibitors. CDKs have been shown to be required for replication of herpes simplex virus (HSV) (Schang, L. M. et al., J. Virol., 72:5626 (1998)). Synovial tissue hyperplasia plays important roles in the development of rheumatoid arthritis; inhibition of synovial tissue proliferation may suppress inflammation and prevent joint destruction. It has been shown that over-expression of CDK inhibitor protein p16 inhibited synovial fibroblast growth (Taniguchi, K. et al., Nat. Med., 5:760-767 (1999)) and joint swelling was substantially inhibited in animal arthritis models.
Selective inhibitors of some CDKs may also be used to protect normal untransformed cells by inhibiting specific phases of cell cycle progression (Chen, et al., J. Natl. Cancer Institute, 92:1999-2008 (2000)). Pre-treatment with a selective CDK inhibitor prior to the use of a cytotoxic agent that inhibits a different phase of the cell cycle may reduce the side effects associated with the cytotoxic chemotherapy and possibly increase the therapeutic window. It has been shown that induction of cellular protein inhibitors of CDKs (p16, p27 and p21) conferred strong resistance to paclitaxel- or cisplatin-mediated cytotoxicity on the inhibitor-responsive cells but not on the inhibitor-unresponsive cells (Schmidt, M, Oncogene, 2001 20:6164-71).
CDK4 and CDK6 are two functionally indistinguishable cyclin D dependent kinases. They are widely expressed with high levels of expression observed in cells of hematopoeitic lineage (CDK4/6 will be used throughout this document to reference both CDK4 and CDK6). CDK4/6 promotes G1-S transition of the cell cycle by phosphorylating the retinoblastoma protein (Rb). CDK4 and CDK6 single knockout mice are viable and double knockout mice die around birth with defective hematopoiesis (Satyanarayana, A. et al., Oncogene, 28:2925-39 (2009); Malumbres, M. et al., Cell, 118:493-504 (2004)). Strong evidence supports a significant involvement of the cyclin D-CDK4-p16INK4A-Rb pathway in cancer development (Malumbres, M. et al., Nature Rev. Cancer, 1:222-31 (2001)). Rb negatively regulates the cell cycle at G1 by sequestering E2F proteins that are required for initiation of S phase. p16INK4A is a key member of the INK4 family of CDK4/6 cellular inhibitors. The genes for Rb and p16INK4A are tumor suppressors that are often deleted or silenced in cancer cells. Additionally CDK4, CDK6 and cyclin D are reported to be amplified in hematologic malignancies and solid tumors. The importance of this pathway in oncogenesis is further supported by the finding that depletion or inactivation of CDK4 inhibits tumor growth in mouse tumor models (Yu, Q. et al., Cancer Cell, 9:23-32 (2006); Puyol, M. Cancer Cell, 18:63-73 (2010)). Rb and p16INK4A are rarely deleted in AML. However, the p15INK4B gene, another member of the INK4 family, has been reported to be down regulated by hypermethylation in up to 60% of AML (Naofumi, M. et al., Leukemia Res., 29:557-64 (2005); Drexler, H. G. Leukemia, 12:845-59 (1998); Herman, J. G. et al., Cancer Res., 57:837-41 (1997)), suggesting a possible critical role for CDK4/6 in AML cells.
FLT3 (Fms-like tyrosine kinase 3, FLK2) is a class III receptor tyrosine kinase. It is activated by the FLT3 ligand (FL) and signals through the PI3K, RAS, and JAK/STAT pathways (Scholl C. et al., Semin. Oncol., 35:336-45 (2008); Meshinchi S. et al., Clin. Cancer Res., 15:4263-9 (2009)). FLT3 plays a role in early hematopoiesis and FLT3 deficient mice have reduced numbers of progenitors of multiple lymphoid lineages (Mackarehtschian K, et al., Immunity, 3:147-61 (1995). Activating mutations in FLT3 are found in approximately 30% of AML patients, representing the most frequent genetic alteration in the disease. About 75% of the activating mutations are internal tandem duplications (ITD) and 25% are point mutations in the activation loop of the kinase domain. The most frequently identified activating point mutation is D835Y (Yamamoto et al., Blood, 97(8): 2434-2439 (2001)). However, mutations have also been found at N841I (Jiang, J. et al., Blood, 104(6): 1855-1858 (2004)) and Y842C (Kindler et al., Blood, 105(1): 335-340 (2005)). Additional point mutations have been identified in the juxtamembrane domain and kinase domain, although these have been shown to result in lower transforming potential (Reindel et al., Blood 107(9): 3700-3707 (2006)).
Murine bone marrow transplanted with a retrovirus expressing the FLT3-ITD has been shown to result in the production of a lethal myeloproliferative disease in mice (Kelly et al., Blood 99: 310-318 (2002)) characterized by leukocytosis consisting of mature neutrophils. This disease did not show a block in differentiation as seen in human AML suggesting that FLT3 mutations confer a proliferative or survival advantage to the cells. Additional oncogene mutation producing a block in differentiation such as AML1/ETO is hypothesized to be required to produce disease that is more similar to human AML.
A number of FLT3 inhibitors have been tested in clinical trials. Although they have shown initial clinical responses in AML, the responses observed were transient and resistance can develop rapidly (Weisberg, E. et al., Oncogene, 29:5120-34 (2010)). The major resistance mechanism appears to be through the acquisition of secondary mutations in FLT3, which may interfere with the binding of FLT3 inhibitors to the FLT3 receptor (Weisberg, E. et al., Oncogene, 29:5120-34 (2010); Chu, S. H. et al., Drug Resist. Update, 12:8-16 (2009)). One such resistance mutation (N676K) was identified in a patient at the time of clinical relapse while on multi-kinase FLT3 inhibitor midostaurin (PKC412) monotherapy (Heidel, F. et al., Blood, 107:293-300 (2006)). Combinations of FLT3 inhibitors with chemotherapy are being tested in clinical trials despite the recognition that chemotherapy is poorly tolerated. Additional possible mechanisms for lack of durable responses include inadequate target coverage (Pratz, K. W., et al., Blood, 139:3938-46 (2009)) and protection of AML cells in the bone marrow where stromal growth factors may provide proliferative signals in addition to FLT3 activation (Tam, W. F. et al., Best Pract. Res. Clin. Haematol., 21:13-20 (2008)). Inhibitors with combined FLT3 and CDK4/6 inhibitory activities are novel and may prove beneficial in treating various cancers including, but not limited to, AML.
Fused tricyclic pyridine, pyrimidine, and triazine compounds useful for treating diseases mediated by CDK4 are disclosed in WO 2009/085185, published on Jul. 9, 2009, which is hereby incorporated by reference in its entirety and for all purposes as if fully set forth herein. Various gem-disubstituted and spirocyclic compounds useful for treating diseases mediated by CDK4 are disclosed in WO 2009/0126584, published on Oct. 15, 2009, which is hereby incorporated by reference in its entirety and for all purposes as if fully set forth herein.
A continued need exists for new compounds that can be used to modulate CDK4, CDK6, and/or FLT3 and can be used to treat various disease conditions associated with these kinases. The compounds of the present invention provide significant improvements in inhibition in one or more of these kinases and have properties making them excellent therapeutic candidates.